Neural regulation of tear production and in particular of lacrimal gland secretion is critical to a healthy ocular surface. Parasympathetic and sympathetic neurotransmitters induce lacrimal gland secretion of protein, electrolytes, and water by using three distinct signaling pathways. In spite of the multitude of pathways, with aging and in Sjogren's syndrome, lacrimal gland secretion fails and dry eye results. In dry eye is a lymphocyte-mediated inflammation blocks neurally stimulated secretion by the lacrimal gland. Determination of the alterations in the cellular signaling pathways that account for the loss of secretion depends upon knowledge of the normal neural signaling pathways. The long-term objective is to investigate the types of purinergic receptors activated by nucleotides in the lacrimal gland, evaluate receptor function, and determine if their dysfunction contributes to the loss of secretion that accompanies inflammation that develops with aging and in Sjogren's syndrome. The following specific aims will be investigated: 1. How does extracellular ATP activate P2X7 receptors and stimulate protein secretion in the lacrimal gland? 2. Do cholinergic and alpha 1-adrenergic agonists release ATP in the lacrimal gland, is ATP released from nerves, myoepithelial cells, or acinar cells, and which signaling pathways does ATP activate in acinar cells? and 3. Does prolonged stimulation of P2X7 receptors activate second messengers that induce pore formation, IL-1beta release, and/or cell death and does this play a role in the secretory failure or cell death that occurs in a mouse model of lacrimal gland dysfunction? The presence of P2X7 receptors will be investigated by RT- PCR, western blotting and immunofluorescence microscopy in acinar cells from rat lacrimal glands. The function of these receptors will be determined by measuring intracellular [Ca2+] and protein secretion. The mechanism and site of cholinergic and alphal-adrenergic agonist release of ATP will be assessed in tissue pieces, myoepithelial cells, and acinar cells by enzymatic assay. The effect of prolonged activation of P2X7 receptors will be determined by measuring pore formation, interleukin 1beta release, and cell death. P2X7 receptor knockout mice and a mouse model of Sjogren's syndrome will be used. Knowledge about the molecules inside lacrimal gland cells that cause secretion will be critical to the development of new drugs to produce tear secretion in individuals who have dry eye resulting from aging or Sjogren's syndrome. [unreadable] [unreadable] [unreadable] [unreadable]